Process for the purification of melphalan

ABSTRACT

A method for the purification and preparation of melphalan that allows to obtain melphalan with purity higher than 99.5% is described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 14/890,540,filed Nov. 11, 2015 which is a 371 of PCT/EP2014/060987, filed May 27,2014, which claims the benefit of Italian Patent ApplicationM12013A000896, filed May 31, 2013, the disclosure of each of which isincorporated by reference.

FIELD OF INVENTION

The present invention relates to a method for the purification ofmelphalan. Melphalan is the L enantiomer of4-[bis(2-chloroethyl)amino]phenylalanine of formula I:

This compound is also known with the name of L-PAM, L-sarcolysine,NCS-8806, CB3025.

Melphalan is commercially known with the name of Alkeran™ in the form oftablets or injectable preparations.

Melphalan is a compound known for its antitumor properties; its Denantiomer and the racemic mixture also have antitumor activity but ofsmall degree.

Melphalan is particularly used in the treatment of multiple myeloma andovarian cancer.

Melphalan is synthesized according to the processes disclosed in U.S.Pat. No. 3,032,584 and U.S. Pat. No. 3,032,585 starting fromDL-phenylalanine with a process comprising the following steps:

-   -   1. nitration in the presence of nitric acid and sulfuric acid;    -   2. protection of the glycine amino group as acetyl, formyl or        phthtaloyl derivative followed by esterification of the carboxy        group;    -   3. reduction of the nitro group to amine by hydrogenation;    -   4. hydroxyethylation reaction of the amine on the aromatic ring        in the presence of ethylene oxide;    -   5. chlorination in the presence of POCl₃ or SOCl₂;    -   6. resolution of the racemic mixture and isolation of        L-enantiomer;    -   7. deprotection of the glycine amino group and hydrolysis of the        ester. RO 57195 discloses the isolation of melphalan        hydrochloride by adding diethyl ether to the aqueous solution        containing it, followed by the addition of Na₂CO₃ or NaOAc to        bring the pH to 0.5 and subsequently to 1.5-2. The purification        is carried out by dissolving in HCl melphalan hydrochloride        obtained by the treatment at pH=2. GB 750,155 discloses the        synthesis of melphalan starting from        4-[bis(hydroxyethyl)amino]phenylalanine ethyl ester by reaction        with POCl₃ or SOCl₂ in the optional presence of an inert solvent        to give the chlorine derivative followed by a reaction with HCl        to remove the protective groups.

GB 783,292 discloses the synthesis of melphalan starting from its nonoptically active precursors by resolution of some intermediates of thesynthesis as brucine salts. It is particularly disclosed the separationof brucine diastereomeric salts of N-acetyl-4-nitro-DL-phenylalanine.The L-isomer, suitably purified from the residues of brucine, ishydrolysed to give 4-nitro-phenylalanine, followed by the esterificationwith phthalic anhydride, the reduction of the nitro group, the reactionof the resultant amine with ethylene oxide, the reaction with POCl₃ orSOCl₂ to obtain the chlorine derivative, the removal of the phthaloylprotective group and of the ethyl ester to give4-bis-(2-chloroethyl)-aminophenylalanine.

CN 101100440 discloses a process for the preparation of melphalanhydrochloride comprising:

-   -   the esterification of the carboxyl group of 4-nitrophenylalanine        in EtOH    -   the protection of the amine in the presence of TEA to give the        N-Boc derivative    -   the hydrogenation of the nitro group    -   the hydroxyethylation reaction in the presence of ethylene oxide    -   the chlorination reaction of the OH groups    -   the deprotection reaction of the amino group    -   the hydrolysis reaction with 2M-6M hydrochloric acid to remove        the protective groups and to obtain the final compound.

EP 0 317 281 discloses the preparation of melphalan hydrochloride byreaction of the N-phthaloyl derivative with ethylene oxide, followed bychlorination, hydrolysis and subsequent formation of melphalanhydrochloride.

WO 2009/117164 discloses a process for the synthesis of melphalancharacterized by the fact that the hydroxyethylation reaction of thearomatic NH₂ group is carried out without the need to protect theglycine NH₂ group.

US 2012/0190887 discloses a process for the preparation ofpharmaceutical grade melphalan hydrochloride comprising a purificationstep by dissolving in hydrochloric acid, adding active charcoal, addingalkali hydroxide, filtering and washing with deionized water, slurryingwith isopropyl ether.

US 2012/0116117 discloses a process for the preparation of melphalanhydrochloride with HPLC purity>99% i.e. conforming to drug regulationspecifications.

In the processes described in the state of the art, melphalan isisolated and purified through some steps that provide a precipitation ofmelphalan in water saturated with sodium acetate at pH=7 followed by oneor more crystallizations from methanol or a precipitation in thepresence of diethylamine at pH=7, followed by washing with methanol.

Most of the purification methods disclosed in the state of the art needto bring the pH to neutrality after the treatment with aqueoushydrochloric acid necessary for the removal of the amine and carboxyprotective groups, to allow the precipitation of melphalan.

These purification methods yield melphalan with a purity from 96% to99%.

The precipitation of melphalan at pH=7, according to the processes knownin the state of the art, leads to the formation of a highly unstablesolid which is particularly hard to filter because of its sponginess.The instability of said spongy solid leads to the formation of animpurity, called “dimer” (impurityG-4-[[2-[[4-[bis(2-chloroethyl)amino]-L-phenylalanine]oxy]ethyl]-(2-chloroethyl)amino]-L-phenylalanine)according to European Pharmacopeia 2012, pages 4658-4659). The limitvalues of such impurity, reported into the European Pharmacopeia, arehigh (limit NMT 1.0%) just because of the difficult in the removal ofthe “dimer” in the process for the production of melphalan.

SUMMARY OF INVENTION

We have now found a method for the purification of melphalan that allowsto obtain melphalan with much higher purity than that required by theEuropean Pharmacopeia, particularly with a purity higher than 99.5%,preferably higher than 99.8%, in which each single impurity identifiedby the European Pharmacopeia is NMT 0.5%, preferably NMT 0.2%.Particularly, the impurity G is preferably NMT 0.2%, most preferably NMT0.1%.

Melphalan obtained after the treatment at pH=2 in the presence of water,methyl-tert-butyl ether and diethylamine is characterized by the absenceof ionizable chlorides by HPLC analysis and will be herein afterindicated as “melphalan base”.

DETAILED DESCRIPTION OF THE INVENTION

It is therefore an object of the present invention a process for thepurification of melphalan comprising:

-   -   (a) the treatment of a compound of formula IV

-   -   wherein R is hydrogen or a linear or branched C₁-C₆ alkyl group,        preferably selected among ethyl, methyl, propyl, most preferably        ethyl and R¹ is hydrogen or an amine protective group,        preferably selected among phthaloyl or tert-butoxycarbonyl, more        preferably tert-butoxycarbonyl; or a hydrochloric salt or a        dimeric form thereof, in HCl 37% under reflux;    -   (b) the isolation of the compound obtained from step (a) in the        presence of water, methyl-tert-butyl ether and diethylamine at        pH=2.

The process for the purification object of the present invention isfollowed by washing the compound obtained from step (b) with a linear orbranched C₁-C₄ alcohol, preferably selected among methanol, ethanol andisopropanol, more preferably ethanol. Said washing allows to remove thediethylammonium salts obtained from treatment (b). The product obtainedby said washing can be stored as such at low temperatures to avoid theformation of dimerization products or can be salified with gaseous HClwhich is added as an ethanol solution in acetone to give melphalanhydrochloride.

The process for the purification object of the present invention allowsto obtain melphalan with a HPLC purity greater than 99.5%, preferablygreater than 99.8%. The step (a) of the process object of the presentinvention is preferably carried out under reflux for at least 16 hours.The ratio HCl 37%/melphalan (titrated) is preferably of about 4:1.

The step (b) is preferably taken at a temperature from 0° C. to 10° C.

The ratio water/melphalan (titrated) is preferably of about 10:1, theratio methyl-tert-butyl ether/water is of about 2:1. In step (b) of theprocess object of the present invention melphalan with a HPLC puritygreater than 99.5%, preferably greater than 99.8%, is obtained.

The method for the purification object of the present inventioncomprises the treatment of melphalan or one of its derivative of formulaIV optionally in a protective and/or salified form, or as a “dimer”,both as a product or an isolated intermediate of the synthesis, both asa reaction crude, in the presence of HCl 37% under reflux, followed bythe treatment at pH=2 of the resultant compound.

The treatment with HCl 37% under reflux allows to obtain melphalandissolved in solution with a purity greater than 99.5%, preferablygreater than 99.8%. Said treatment allows to hydrolyze any protectivegroups on the molecule and to purify, at the same time, the resultantproduct by hydrolysis of any impurities in the reaction medium such forexample esters, dimers, trimers of melphalan.

The isolation of the product after the treatment with HCl 37% underreflux is carried out by treatment at pH=2 in the presence ofdiethylamine and methyl-tert-butyl ether. The isolated product ischaracterized by the absence of ionizable chlorides and by a low abilityto form undesirable by-products, such as for example the “dimer”(impurity G).

The process for the purification object of the present invention can beused to obtain melphalan with a high purity (greater than 99.5%) bothstarting from a reaction crude and from melphalan with a low puritydegree or from a derivative of formula IV thereof.

In a preferred embodiment the process for the purification object of thepresent invention is carried out at the end of a process of synthesis ofmelphalan which comprises:

-   -   1. the alkylation reaction of a compound of formula II

-   -   wherein R² is a linear or branched C₁-C₆ alkyl group, preferably        selected among ethyl, methyl, propyl, most preferably ethyl and        PG is an amine protective group, preferably selected among        phthaloyl or tert-butoxycarbonyl group, most preferably        tert-butoxycarbonyl;    -   in the presence of an aprotic polar solvent, a base and an        alkylating agent, preferably selected among iodoethanol,        chloroethanol or ethylene oxide to give a compound of formula        III

-   -   wherein R² and PG have the above reported meanings;    -   2.the chlorination reaction of the resultant compound of formula        III in the presence of a chlorinating agent preferably selected        among POCl₃ and SOCl₂ in a suitable solvent preferably selected        among isopropylacetate or ethylacetate, preferably        isopropylacetate to give a compound of formula IV-1

-   -   wherein R² and PG have the above reported meanings.

It is therefore a further object of the present invention a process forthe preparation of melphalan comprising:

-   -   1. the alkylation reaction of a compound of formula II

-   -   wherein R² is a linear or branched C₁-C₆ alkyl group, preferably        selected among ethyl, methyl, propyl, most preferably ethyl and        PG is an amine protective group, preferably selected among        phthaloyl or tert-butoxycarbonyl group, most preferably        tert-butoxycarbonyl;    -   In the presence of an aprotic polar solvent, a base and an        alkylating agent, preferably selected among iodoethanol and        chloroethanol or ethylene oxide to give a compound of formula        III

-   -   wherein R² and PG have the above reported meanings;    -   2.the chlorination reaction of the resultant compound of formula        III in the presence of a chlorinating agent preferably selected        among POCl₃ and SOCl₂ in a suitable solvent preferably selected        among isopropylacetate or ethylacetate, preferably        isopropylacetate to give a compound of formula IV-1

-   -   wherein R² and PG have the above reported meanings;    -   3.the isolation and purification of melphalan comprising:        -   (a) the treatment of a compound of formula IV-1 in HCl 37%            under reflux;        -   (b) the isolation of the compound obtained from step (a) in            the presence of water, methyl-tert-butyl ether and            dietylamine at pH=2;        -   (c) the washing of the compound obtained from step (b) with            a linear or branched C₁-C₄ alcohol, preferably selected            among methanol, ethanol, isopropanol, most preferably            ethanol;    -   4.the optional treatment of the compound obtained from step (c)        with a mineral acid, preferably with an ethanol solution of        gaseous HCI in acetone.

In step 1) of the process object of the present invention the aproticpolar solvent is preferably selected among acetonitrile anddichloromethane, acetonitrile is preferably used; the base is preferablyselected among Na₂CO₃, K₂CO₃, Cs₂CO₃, CaO, Na₂CO₃ is most preferablyused.

In step 2) of the process object of the present invention POCl₃ inisopropylacetate is preferably used.

Preferably in step 4) of the process object of the present invention anethanol solution of gaseous HCl in acetone is used in a concentration offrom about 16% to about 25%.

A further preferred embodiment of the process object of the presentinvention consists of the synthesis and purification of melphalancomprising:

-   -   1) the alkylation reaction of the amine group on the aromatic        ring of N-BOC-L-phenylalanine ethyl ester in the presence of        acetonitrile, Na₂CO₃ and iodoethanol to give        4-(bis(2-hydroxyethyl)-amino-N-BOC-L-phenylalanine ethyl ester;    -   2) the chlorination reaction of the compound obtained in step 1)        in the presence of POCl₃ and isopropylacetate;    -   3) the isolation and purification of melphalan comprising:        -   (a) the treatment of the compound obtained in step 2) in the            presence of HCl 37% under reflux;        -   (b) the isolation of the compound obtained in step a) with            water, methyl-tert-butyl ether and diethylamine at pH=2;        -   (c) the washing of the compound obtained in step b) with            ethanol;

4) the treatment of the compound obtained in step c) with an ethanolsolution of HCl in acetone

Melphalan and its hydrochloride salt having HPLC purity >99.5%,preferably >99.8% are a further object of the present invention.

The process object of the present invention allows to obtain melphalanwith a HPLC purity greater than the processes known in the state of art.Particularly, the hydrolysis reaction reported in step a) allows toobtain high purities through the removal of further impurities in themixture such as for example dimers, trimers, esters etc.

The purity result is surprisingly obtained only by using HCl 37% underreflux. In fact, using HCl at different concentrations, for example at32%, melphalan with purity greater than 99.5% is not obtained whileoperating under the same conditions of the purification processaccording to the invention.

The subsequent isolation at pH=2 in step (b) surprisingly allows toobtain a compound with a high purity and characterized by a highstability in comparison to the spongy solid isolated under the neutralconditions described in the known art. The greater stability is probablygiven by a less capability to form the “dimer” (impurity G) compared tomelphalan obtained according to the methods known in the state of art.

The washing of the compound reported in step (c) with ethanol allows toremove diethylammonium chloride salts, residues of step (b).

All the terms used in the present application, unless otherwiseindicated, are to be understood in their common meaning as known in theart. Other more specific definitions for certain terms, as indicated inthis application, are underlined later and are constantly applied forthe whole description and the claims unless a different definitionprovides specifically a wider meaning.

The term “polar solvent” relates to a solvent which behave as a protondonor, such as water; an alcohol, for example, methanol, ethanol,propanol, iso-propanol, butanol, tert-butanol; or a polarized solvent,such as esters, for example ethyl acetate, butyl acetate, nitriles, forexample acetonitrile; ethers, for example, tetrahydrofuran, dioxane;ketones, for example, acetone, methyl butyl ketone; and the like.

Further information about solvent can be found in organic chemistrybooks or in specialized monographs, for example Organic SolventsPhysical Properties and Methods of Purification, 4th ed., John A.Riddik,et al., Vol II, in “Techniques of Chemistry Series”, John Wiley & Sons,NY, 1986. Such solvents are known to the person skilled in the art andit is clear to the person skilled in the art that different solvents andmixtures thereof can be preferred, depending on the specific compoundsand on the reaction conditions, being their choice influenced, forexample, by solubility and reagent reactivity, by preferred temperatureranges. Although the invention has been described in its characterizingfeatures, the equivalents and modifications obvious to the skilled inthe art are included in the following invention.

The present invention will now be illustrated by some examples whichhave not to be seen as limiting the scope of the invention.

EXAMPLE 1 Preparation of4-(bis-(2-hydroxyethyl)-amino-N-BOC-L-phenylalanine ethyl ester

Into a suitable reactor A, 2.0 kg of N-BOC-L-phenylalanine ethyl esterand acetonitrile were loaded. The suspension was let under stirringuntil complete dissolution, maintaining the temperature at 23° C. and1.5 kg of sodium carbonate were then added. Subsequently, 3.2 L of2-iodoethanol were added dropwise, the solution was heated up to 85° C.and these conditions were maintained for 2 hours. At the end of thereaction the mixture was quickly cooled down to 20° C.

In another suitable reactor B, 35L of water and 4 kg of Celite® wereloaded and the mixture was left under stirring for at least 5 minutes.The compound obtained into reactor A was transferred into reactor B andthe suspension was kept under stirring for an hour. The suspension wasfiltered and washed with 12L of water.

The wet Celite® obtained into the reactor B and 140 L of ethyl acetatewere loaded into a reactor C and the mixture was kept under stirring for30 minutes then filtered by collecting the filtrate into a reactor D andletting the phases separate. The organic phase was distilled at reducedpressure. The residue was purified by chromatography using methylenechloride/ethyl acetate about 2:1.

About 1.8 kg of 4-(bis-(2-hydroxyethyl)-amino-N-BOC-L-phenilalanineethyl ester were then obtained.

EXAMPLE 2 Preparation of Melphalan Hydrochloride

4-(bis-(2-hydroxyethyl)-amino-N-BOC-L-phenilalanine ethyl estercontained into the rich fractions was dissolved in 16 L of isopropylacetate and dropped into 4.6 L of phosphoryl chloride, maintaining thetemperature at 40° C., in an inert atmosphere. The solution was heatedat 50° C. and the solution was kept under stirring for 2 hours and 30minutes in an inert atmosphere.

At the end of the reaction the solution was then transferred into aseparated funnel letting the phases separate for 30 minutes.

The phase containing the chlorinated product was dropped into 8.8 L of37% HCl and let under stirring at 130° C. under reflux for at least 12hours. The solution was subsequently cooled down to 50° C.

At the end of the reaction the solution was cooled down to 20° C. understirring and subsequently concentrated at a reduced pressure.

To the concentrated residue were added 9 L of water, 18 L ofmethyl-tert-butyl ether and 2.6 L of diethylamine. The mixture was keptunder stirring up to stable pH=2 and these conditions were maintainedfor 10 minutes, the mixture was subsequently filtered and washed with 10L of methyl-t-butyl ether.

The resultant product was transferred into a suitable reactor and 18 Lof ethanol at 99.9% were subsequently loaded. The suspension was leftunder stirring for at least 10 minutes, then filtered. The product wasdried under vacuum for 8-12 hours obtaining melphalan.

Into a suitable reactor were loaded 0.7 Kg of melphalan, obtained aspreviously described, and 22 L of acetone, the solution was cooled downand kept under stirring for 30 minutes. The stoichiometric amount ofgaseous HCl absorbed in ethanol was subsequently added according to thetitration. The suspension was kept under stirring under cooling for afew hours. The suspension was filtered and the solid washed with acetoneand methyl-tert-butyl ether.

The solid was dried under vacuum for one night.

About 0.7 kg of melphalan hydrochloride were obtained.

1) Melphalan and melphalan hydrochloride having HPLC purity of >99.5%.2) Melphalan and melphalan hydrochloride according to claim 1 whereinimpurity G is NMT 0.5%. 3) Melphalan and melphalan hydrochlorideaccording to claim 1 having HPLC purity of >99.8%. 4) Melphalan andmelphalan hydrochloride according to claim 1 wherein impurity G is NMT0.2%. 5) Melphalan and melphalan hydrochloride according to claim 1wherein impurity G is NMT 0.1%.